WO2017165800A2 - Nanocapteurs et procédés pour la détection de marqueurs biologiques - Google Patents

Nanocapteurs et procédés pour la détection de marqueurs biologiques Download PDF

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WO2017165800A2
WO2017165800A2 PCT/US2017/024070 US2017024070W WO2017165800A2 WO 2017165800 A2 WO2017165800 A2 WO 2017165800A2 US 2017024070 W US2017024070 W US 2017024070W WO 2017165800 A2 WO2017165800 A2 WO 2017165800A2
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particle
nanosensor
sample
protein
proteins
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WO2017165800A3 (fr
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Massoud Motamedi
Allan R. Brasier
Stefan H. Bossmann
Christopher T. Culbertson
Deryl Troyer
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The Board Of Regents Of The University Of Texas System
Kansas State University Research Foundation
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Priority to US16/088,407 priority Critical patent/US11965881B2/en
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Publication of WO2017165800A3 publication Critical patent/WO2017165800A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to nanosensors and instruments (e.g., microfluidics and optical sensors) and detection techniques (e.g., multiplexing) for detecting target biological markers in a specimen collected from a subject.
  • nanosensors and instruments e.g., microfluidics and optical sensors
  • detection techniques e.g., multiplexing
  • Inflammation is a protective response of the body to cellular damage and tissue injury. Under normal and controlled conditions, the role of this process is to remove the injurious agents while promoting wound healing and repair. However, under uncontrolled conditions, inflammatory responses can lead to excessive cellular damage resulting in chronic inflammation and destruction of normal tissue. Inflammatory airway and lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), or lung injury are characterized by chronic inflammation, fibrosis, and airway remodeling. Asthma affects the health of 26 million people, including 7 million children in the United States. It is characterized by repeated episodes of wheezing, breathlessness, chest tightness, and nighttime or early morning coughing.
  • COPD chronic obstructive pulmonary disease
  • RSV infections In both children and adults, viral infections trigger asthma exacerbations by stimulating inflammatory gene expression programs in infected epithelium. These viruses include Respiratory Syncytial Virus (RSV)/human metapneumoviruses in children and Rhinovirus in adults.
  • RSV infections of the small airways (bronchioles) is the most common cause of lower respiratory tract infections in children, infecting virtually all children by the age of 3.
  • a recent prospective, population-based study of 5000 children presenting for acute medical care estimated that 18% of acutely ill children have acute RSV infection.
  • the presence of RSV infection produces 3-times the risk of subsequent hospitalization over that seen in infections with other common cold viruses, and in young children, hospitalization rates are 17 per 1000 babies.
  • Rhinovirus is the most common cause of viral respiratory tract infections in adults, and is responsible for exacerbations of asthma in this population.
  • Cytokines are small proteins (-5-20 kDa) that are important in cell signaling. Cytokine release has an effect on the behavior of cells around them. Cytokines are also involved in autocrine signaling, paracrine signaling and endocrine signaling as immunomodulating agents.
  • the term "cytokine” includes chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. They are produced by macrophages, B lymphocytes, T lymphocytes as well as endothelial cells, fibroblasts, and various stromal cells. Cytokines modulate the balance between humoral and cell-based immune responses. There are both pro-inflammatory cytokines and anti-inflammatory cytokines.
  • Pro-inflammatory cytokines include IL- ⁇ , Interleukin 6 (IL- 6), and T F- ⁇ , which are involved in the process of pathological pain.
  • Anti-inflammatory cytokines include IL4, IL-10, IL-13 and IFN-alpha.
  • Macrophage Inflammatory Protein- 3 (MTP3A) is another small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes and weakly attracts neutrophils. CCL20 is implicated in the formation and function of mucosal lymphoid tissues.
  • IL-6 acts as both a proinflammatory cytokine and an anti-inflammatory myokine. IL-6 is secreted by T cells and macrophages to stimulate immune response.
  • Thymic stromal lymphopoietin plays an important role in the maturation of T cell populations through activation of antigen presenting cells. Produced mainly by non-hematopoietic cells such as fibroblasts, epithelial cells and different types of stromal or stromal-like cells. Expression is linked to many disease states including asthma, inflammatory arthritis, atopic dermatitis and other allergic states. Further, co-expression of various markers, such as cytokines and MMPs can be used to detect and monitor condition progression. There is a need for techniques and instruments capable of such co-detection, which would greatly improve specificity and the diagnostic power of collected patient specimen.
  • the present invention is broadly concerned with nanosensors, microfluidic devices, and detection techniques for capturing, detecting, and quantifying biological markers in biospecimens collected from a subject (typically but not necessarily a mammalian subject, and usually a human patient, although veterinary uses are contemplated, such as in dogs, cats, horses, pigs, monkeys, rodents, etc.).
  • a subject typically but not necessarily a mammalian subject, and usually a human patient, although veterinary uses are contemplated, such as in dogs, cats, horses, pigs, monkeys, rodents, etc.
  • microfluidic detection of a biological marker in a biological sample collected from a subject generally comprise analyzing a biological sample with a microfluidics device.
  • the microfluidic devices comprise a cartridge and a planar substrate retained in the cartridge.
  • the cartridge comprises a sample inlet well formed in the cartridge above a sample application region in or on the substrate and at least one detection region in fluid communication with the sample application region via a microfluidic channel extending from the sample application region to the detection region.
  • the microfluidic channel has a terminal end positioned distal from the inlet well, and an optional absorbent pad positioned at the terminal end (to facilitate flow of the sample completely through the channel).
  • the detection region comprises a first nanosensor immobilized therein or thereon.
  • the nanosensor comprises a central carrier particle, a first particle tethered to the carrier particle via an oligopeptide linkage, and a second particle directly attached to the carrier particle, wherein at least one of the first and second particles is a detectable particle and the other of the first and second particles is a quencher particle.
  • the oligopeptide linkage comprises a supramolecular recognition sequence, a protease consensus sequence, a post-translationally modifiable sequence, or a sterically hindered benzylether bond for specific interaction with the target biological marker.
  • the biological sample collected from the subject is introduced into the inlet well and the biological sample flows to the terminal end along the microfluidic chamber, wherein the biological sample contacts the detection region before reaching the terminal end.
  • the detection region is exposed to an energy source to generate a detectable signal from the detectable particle in the first nanosensor, and changes (e.g., decrease or increase in signal, appearance of new signal, etc.) in the detectable signal are detected based upon interaction of the biological sample (and more specifically the target marker, if present in the sample) with the first nanosensor.
  • the microfluidic devices general comprise a cartridge and a planar substrate retained in the cartridge.
  • the cartridge comprises a sample inlet well formed in the cartridge above a sample application region in or on the substrate and at least one detection region in fluid communication with the sample application region via a microfluidic channel extending from the sample application region to the detection region.
  • the microfluidic channel has a terminal end positioned distal from the inlet well, and an optional absorbent pad positioned at the terminal end (to facilitate flow of the sample completely through the channel).
  • the detection region comprises a nanosensor immobilized therein or thereon.
  • the nanosensor comprises a central carrier particle, a first particle tethered to the carrier particle via an oligopeptide linkage, and a second particle directly attached to the carrier particle, wherein at least one of the first and second particles is a detectable particle and the other of the first and second particles is a quencher particle.
  • the oligopeptide linkage comprises a supramolecular recognition sequence, a protease consensus sequence, a post-translationally modifiable sequence, or a sterically hindered benzylether bond for specific interaction with the target biological marker.
  • nanosensors for detecting the presence of an active protein in a sample.
  • the nanosensors comprise a central carrier particle and a first particle connected to the central carrier particle via an oligopeptide linkage that comprises a supramolecular recognition sequence, wherein the protein has specific binding to the supramolecular recognition sequence without chemical modification or enzymatic cleavage of the linkage.
  • a second particle is also directly attached to the central carrier particle via a non-cleavable linkage (that is generally shorter than the oligopeptide linkage of the first particle).
  • at least one of the first and second particles is a detectable particle and the other of the first and second particles is a quencher particle.
  • the nanosensor is configured such that the detectable particle and quencher particle are separated by a distance that enables Forster resonance energy transfer.
  • the methods generally comprise contacting a biological sample from the mammal with a nanosensor described herein; exposing the nanosensor to an energy source to generate a detectable signal from the detectable particle; and detecting changes in the detectable particle signal upon contact of the nanosensor with the sample (and more specifically with active proteins, if present, in the sample) wherein the changes correspond to protein activity in the sample.
  • Figure (Fig.) 1 is a cartoon illustration of an example of a cytokine nanosensor in accordance with an embodiment of the invention
  • Fig. 2 is a cartoon illustration of the mechanism of action of an example of a cytokine nanosensor in accordance with an embodiment of the invention
  • Fig. 3 is a cartoon illustration of an inverse nanosensor in accordance with an embodiment of the invention, in which the detectable particle is directly attached to the central carrier particle (e.g., via short dopamine spacer or designer polymer), while the quencher particle is tethered via a cleavable linkage;
  • Fig. 4 is a cartoon illustration of different nanosensor types that can be used in the microfluidics devices, including (A) protease sensors; (B) arginase sensors; and (C) cytokine sensors;
  • Fig. 5 is an illustration of a nanosensor using a starburst dendrimer containing gold nanoparticles in accordance with embodiments of the invention
  • Fig. 6 is a cartoon illustration of the chemistry for attaching the nanosensors to a cellulose substrate in a microfluidics device in accordance with embodiments of the invention
  • Fig. 7 illustrates a (A) top-down view and (B) side view of a generalized design for a microfluidics device in accordance with embodiments of the invention
  • Fig. 8 is a flow chart of a learning algorithm for correlating data captured with the microfluidic devices
  • Fig. 9 is a graph of the calibration results for the nanosensors for cytokine detection, showing the log [cytokine concentration] vs. integrated fluorescence intensity, with a maximal experimental error from 5 repetitions of +/- 3 relative percent;
  • Fig. 10 is a graph of the MMP9, E, MIP-1, and Granzyme B levels detected using a
  • Fig. 11 is a graph of measurements of MMP8, neutrophil elastase, granzyme B and MCP- 1 levels in murine BALF from Example 5 captured using a Synergy HI fluorescence plate reader;
  • Fig. 12 is a graph of the hydrodynamic diameters of (forming) aggregates between the exosomes in BALF and the nanosensors designed for targeting CD9, CD63, and CD81.
  • the Figure shows Hydrodynamic Diameter vs. Time (in minutes). The experiments were performed using a Nanobrook 90Plus Zeta Particle Size Analyzer (Brookhaven Instruments Corporation);
  • Fig. 14 illustrates the general Configuration of IDEA valves.
  • A COMSOL multiphysics simulation 2-D cross section of an IDEA actuator.
  • B Depiction of the operation of an IDEA actuator.
  • the switch e.g. optodiode
  • the force generated between the two electrodes closes the fluidic channel between them.
  • the bottom PDMS layer may be replaced by a thin layer high dielectric film. This film can be made amenable to surface modification;
  • Fig. 15 illustrates 3 valves along a channel that can be configured to create a peristaltic pump in the microfluidic device
  • Fig. 16 is a diagram (not to scale) of a point of care device with integrated IDEAs for the detection of biomarkers in bodily fluids;
  • Fig. 17 is a graph showing (left panel) the differential expression of secreted proteins (top) vs cell lysates (bottom) for hBECS, and (right panel) the differential expression of secreted proteins in secreted proteins vs lysates for hSAECs;
  • Fig. 19 shows graphs of SID-SRM for each protein providing independent confirmation of differential secreted proteins
  • Fig. 20 shows graphs CCL20, TSLP and CCL3-L1 are selectively secreted by lower hSAECs.
  • SID-SRM-MS for each protein by cell type from uninfected (-) or RSV infected (+) cells.
  • nanosensors for detecting cytokines, and other proteins based upon supramolecular recognition without chemical modification or enzymatic cleavage.
  • an oligopeptide tether between a central carrier particle and a detectable particle is recognized by the target protein, which binds thereto. Binding physically extends the recognition sequence linearly, increasing the distance between the detectable particle and the carrier particle, giving rise to a detectable change in the nanosensor, which is indicative of the presence of the target protein.
  • An exemplary depiction of a nanosensor is shown in Fig. 1.
  • nanoplatforms for protease detection such as those described in U.S. Patent No. 8,969,027, incorporated by reference herein to the extent not inconsistent with the present disclosure
  • the particular nanosensors do not involve cutting or severing the oligopeptide between the nanoparticle and dye, or changing the chemical characteristics, but rather a physical binding of the target protein to the recognition sequence.
  • this binding leads to a change in the average distance between two particles, resulting in a characteristic change in a detectable signal for the particles used in the nanosensor, such as a fluorescence spectrum.
  • the observable change in fluorescence is a function of protein activity.
  • the quenching effect can be enhanced by tethering a second dye to the surface of the carrier nanoparticle to facilitate FRET (Forster Resonance Energy Transfer).
  • FRET Form Resonance Energy Transfer
  • the nanosensors according to these embodiments comprise an oligopeptide, which is used as a peptide linkage between two particles.
  • the linkage comprises a supramolecular recognition sequence for the target protein, which means that the linkage sequence has specificity (i.e., contains a selective binding site) for the target protein).
  • the linkage sequences avoid non-specific binding motifs.
  • the oligopeptide linkage containing the supramolecular recognition sequence is preferably "non- cleavable" which means that it does not contain protease cleavage sites, and the like.
  • the oligopeptide linkage attaches a detectable particle to a central carrier particle.
  • a quencher particle is also directly tethered or attached to the central carrier particle, such as through an amide bond.
  • the phrase "directly attached” as used herein means that the quencher particle is connected directly to the surface of the central particle, or to a ligand layer on the surface of the central particle (and is not attached via an enzymatic substrate sequence or other consensus sequence).
  • the oligopeptide linkage in the nanosensor has a folded secondary structure, such as an alpha-helix and/or beta-sheet structure, so that the detectable particle is separated from the central carrier particle (and quencher particle at the carrier particle's surface) by a first distance.
  • the target protein such as a cytokine
  • the protein binds to the supramolecular recognition sequence in the oligopeptide modifying the secondary structure of the oligopeptide sequence, such that it linearly extends or unfolds, and thus changes the distance of the second particle to the central carrier particle (and quencher particle).
  • the detectable particle is then separated from the central carrier particle (and quencher particle) by a second distance that is greater than the first distance in the initial nanosensor.
  • the detectable particle generates a detectable signal that changes depending upon its proximity to the carrier particle or quencher particle, and which can be detected and correlated with activity of the target protein.
  • the central carrier particle can be a variety of particles discussed herein, with the proviso that it can be attached with two or more different particles.
  • the nanosensor comprises a plurality of quencher particles attached to a single central carrier particle, and a plurality of detectable particles attached to the same carrier particle via respective oligopeptide linkages.
  • the amount of quencher particles attached to the carrier particle is greater than the amount of detectable particles tethered to the carrier particle.
  • the ratio of the number of quencher particles (directly attached) to detectable particles (tethered) on each carrier particle is from about 1 : 1 to about 1 : 100, more preferably from about 1 : 1.5 to about 1 :35, and more preferably about 1 :35.
  • the central carrier particle is one that is capable of SET (Dipole-surface Energy Transfer), and/or participates in quenching of the detectable signal of one or both of the attached particles (and particularly the second particle).
  • the central carrier particle does not actively participate in the signal being detected in the assay, but is simply a carrier structure for tethering the first and second particles.
  • Non-plasm onic particles can be used in certain embodiments.
  • the detectable particles generate a detectable signal (e.g., optical or spectroscopic), such as fluorescence or color change, which can be perceived visually or measured with an appropriate instrument.
  • a detectable signal e.g., optical or spectroscopic
  • the quencher and detectable particles are selected to show intense FRET in the pair.
  • the quencher and detectable particles are paired so as to enhance the SET quenching of the carrier particle.
  • the nanosensors are particularly suitable for detection methods based upon surface plasmon resonance and FRET between non-identical particles (i.e., nanoparticles or a dye and porphyrin, or two different dyes).
  • FRET describes energy transfer between two particles.
  • Surface plasmon resonance is used to excite the particles.
  • a donor particle initially in its excited state may transfer this energy to an acceptor particle in close proximity through nonradiative dipole-dipole coupling.
  • a first emission is observed upon excitation of the donor particle. Once the peptide binds to the supramolecular recognition sequence, FRET change is observed, and the emission spectra changes. In some instances, only the donor emission is observed.
  • the emission spectra simply changes (increases or decreases).
  • both particles are within the so-called Forster-di stance, energy transfer occurs between the two particles and a red- shift in emission is observed.
  • the energy of the electronically excited state or surface plasmon of the second particle is at least partially transferred to the first particle.
  • this means that a detectable signal e.g., fluorescence, light
  • a detectable signal e.g., fluorescence, light
  • light is emitted only from the detectable particle and a distinct blue-shift in absorption and emission can be observed. This is because the distance between both particles increases.
  • a signal may be detectable from both particles in the initial nanosensor, however, upon modification of the substrate sequence, the signal intensity from the detectable particle increases as the distance between the two particles increases.
  • the quencher particle (directly attached to the central particle) is an acceptor and the detectable particle (tethered via the oligopeptide) is a donor.
  • excitation of the nanosensor is directed towards the particle having a higher energy state (e.g., the donor particle).
  • Excitation of the detectable particle can preferably performed between about 400 nm and about 1500 nm, more preferably between about 500 nm to about 800 nm, and even more preferably between about 650 nm and about 800 nm.
  • chromophore/luminophore particle pairs there is also preferably an overlap between the excitation spectrum of the first chromophore/luminophore and the fluorescence or phosphorescence spectrum of the second chromophore/luminophore to permit adequate Forster energy transfer.
  • cyanine 5.5 (donor) and cyanine 7.0 (acceptor) form a very attractive FRET -pair.
  • Forster-di stance energy transfer occurs between the quencher and detectable particles and a change in absorbance and/or emission of the nanoplatform is observed.
  • the energy of the electronically excited state or surface plasmon of the detectable particle is at least partially transferred to the quencher particle.
  • Excitation is preferably performed with an energy source of appropriate wavelength selected from the group consisting of a tungsten lamp, laser diode, laser, and bioluminescence (e.g., luciferase, renilla, green fluorescent protein).
  • the changes in absorption and/or emission of the particles as the peptide binds (without cleaving or chemically modifying) the oligopeptide linkages will be observed over a time period of from about 1 second to about 120 minutes, preferably from about 1 second to about 30 minutes, and in some cases from about 30 seconds to about 10 minutes, depending upon protein activity.
  • the assay can first be calibrated for the particular nanosensor using a control sample with and without the target proteins, as described in the working examples.
  • the nanosensors would typically be dispersed into a pharmaceutically acceptable solvent system, which could be any type of suitable diluent, excipient, vehicle or the like.
  • a pharmaceutically acceptable solvent system which could be any type of suitable diluent, excipient, vehicle or the like.
  • pharmaceutically acceptable means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained.
  • a pharmaceutically-acceptable carrier would naturally be selected to minimize any degradation of the nanoplatform other agents and to minimize any adverse side effects in the subject (for in vivo administration), as would be well known to one of skill in the art.
  • Pharmaceutically-acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use, and will depend on the route of administration.
  • compositions suitable for administration via injection are typically solutions in sterile isotonic aqueous buffer.
  • Exemplary carriers include aqueous solutions such as normal (n.) saline (-0.9% NaCl), phosphate buffered saline (PBS), sterile water/distilled autoclaved water (DAW), or other acceptable vehicles, and the like.
  • the nanosensors are particularly suited for detecting cytokines.
  • the oligopeptide linkage comprises a linear supramolecular recognition sequence (i.e., paratope) for the receptor/binding site of the cytokine of interest.
  • a signal change e.g., fluorescence increase
  • the nanosensors can measure cytokine concentrations.
  • this technology can be extended to virtually any protein target of interest.
  • one advantage of the inventive nanosensors is the flexibility to adapt the underlying nanosensor structure by modifying the particles, oligopeptide linkages, and the like to suit the sensor technology available, and likewise, using a variety of sensor technologies for detecting additional targets.
  • a similar nanosensor can be prepared for detecting protease activity, similar to described in U.S. Patent No. 8,969,027, except that a quencher particle is directly attached to the central carrier particle as described herein to enhance the detectable signal, see Fig. 4A.
  • methods described herein can also be adapted for use with other nanosensors, such as those described in WO 2016/149637 for detection of enzymatic posttranslational modification, see Fig. 4B. Exemplary supramolecular recognition sequences are listed in Table 1 below.
  • the supramolecular recognition sequences can include one or more spacer residues on the N- and/or C-terminal ends.
  • spacer residues For example, between 1 and 10 amino acids (any amino acids, naturally and non-naturally, L- and D-, or combinations thereof) can be used at one or both ends as spacers.
  • Examples include N-terminal sequences such as GAG- and C-terminal sequences such as -AG.
  • Any other linear peptide sequence of suitable length (e.g., at least 10 amino acid residues) can be used for the supramolecular recognition sequences, with the exception of sequences featuring consensus motifs. If synthesized on a peptide synthesizer, peptide sequences up to about 25 amino acid residues can be used, whereas sequence synthesized in an organism (e.g., E. coli) can be up to about 300 amino acid residues in length. Any designed sequence should to be checked for the presence of cleavage sequences, utilizing a data bank, such as MEROPS.
  • a data bank such as MEROPS.
  • peptide-aptamers for targeting CD9, CD63, and CD81 which are generally accepted surface markers of exosomes have also been developed. They are capable of binding to the exosomes and permit their isolation directly from collected biospecimens using the nanosensors without prior (ultra)centrifugation.
  • the combined use of CD9, CD63, and CD81 will ensure that virtually all exosomes occurring from the sample, such as from airway cells, will be trapped. After treatment with a lysis buffer, the cargo of the captured exosomes can then be analyzed. Analysis of exosomes presents another existing aspect of the technology. Exosomes occurring from small airway epithelial cells and bronchial airway epithelial cells differ significantly in their proteasomes (including the tetraspanin content (CD9, CD 63, CD81) in their outer membranes.
  • Enzyme consensus sequences that can also be used in nanosensors in combination with one or more above include those in Table 2 below, where the cleavage point is indicated by the
  • inflammatory biological markers be detected along with detection of specific viral, bacterial, or mold infections.
  • nanosensors could be prepared using a peptide aptamer against Capsid B
  • nanosensors for viral infections can be prepared using protease consensus sequences for HIV detection (SAVL-LEAT (SEQ ID NO: 74), or SQNY-PIVQ (SEQ ID NO: 75)).
  • each of the foregoing sensors described above can be designed in an alternative configuration where the detectable particle is attached to the central carrier particle in a "permanent" manner (e.g., via non-cleavable peptide sequence), whereas the quencher particle is attached via a supramolecular recognition sequence, a protease consensus sequence, a post-translationally modifiable sequence, or a sterically hindered benzylether bond that interacts with the target biomarker.
  • the quencher particle while the detectable particle remains relatively stationary and affixed to the carrier particle, the quencher particle is instead either cleaved or moved away from the detectable particle and carrier particle, to effect the change in distance between the particles, as described above.
  • the increase in distance between the quencher particle and the detectable particle results in a detectable change in the signal from the nanosensor.
  • Alternative embodiments of the invention concern "inverse" nanosensors from protease sensors discussed here, and illustrated in Fig. 3.
  • the detectable particle When using a microflui die-based device, such as paper microfluidic devices, the detectable particle will generally be cleaved off when enzymatic activity is detected. However, it would be desirable if the detectable signal occurring from this particle would occur from a constrained site on the test strip. Therefore, we have constructed the "inverse nanoplatforms for protease detection", in which the detectable particle is linked to the carrier particle by a short (e.g., 1.4-1.5nm) peptide sequence.
  • H2S Hydrogen Sulfide
  • COPD chronic obstructive pulmonary disease
  • the nanosensor is utilized for measuring the H2S concentration in biospecimens.
  • HS " is a potent nucleophile that readily reacts with metal centers (for instance in coenzymes).
  • a detectable particle is tethered to a central carrier particle via a cleavable sterically hindered benzyl ether bond (i.e., with adjacent carbons substituted with methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.). Nucleophilic cleavage of the bond releases the detectable particle, and the associated change in the nanosensor can be detected.
  • nanosensors utilized in accordance with the invention will be designed to include a supramolecular recognition sequence, a protease consensus sequence, a post-translationally modifiable sequence, or a sterically hindered benzylether bond that gives rise to a specific interaction with a biological marker.
  • references to "specific interactions" is intended to differentiate the inventive sensors from non-specific binding or reactions between molecules, and means that the set of specific target analytes for which the oligopeptide sequence can interact is limited, and in some cases even exclusive, such that neither binding nor enzymatic cleavage occurs at an appreciable rate with any other molecule.
  • the mechanism for these preferred configurations of nanosensors is illustrated in Fig. 4.
  • a number of different types of particles can be used to form the nanosensors, depending upon the type of sensor used to measure the target's activity, as discussed in more detail below.
  • the excitation and emission spectral maxima of the particles are between 650 and 800 nm.
  • Preferred particles for use in the nanosensors are selected from the group consisting of nanoparticles, chromophores/luminophores, quantum dots, viologens, and combinations thereof.
  • nanoparticle refers to nanocrystalline particles that can optionally be surrounded by a metal and/or nonmetal nanolayer shell.
  • Such nanoparticles can be metal nanoparticles: metal, metal alloy, metal oxide, or core/shell metal nanoparticles (e.g. Fe 2 Cb, Fe30 4 ).
  • Metal nanoparticles can alternatively be surrounded by a second shell of silica, as described in U.S. Patent No. 8,877,951, incorporated by reference herein to the extent not inconsistent with the present disclosure.
  • the optimal distance between plasmonic core and shell can be determined experimentally.
  • Suitable nanoparticles preferably have a diameter of from about 1 nm to about 100 nm, more preferably from about 10 nm to about 50 nm, and even more preferably from about 5 nm to about 20 nm.
  • Metal nanoparticles can comprise any type of metal (including elemental metal) or metal alloy.
  • the metal or metal alloy nanoparticles comprise a metal selected from the group consisting of gold (Au), silver (Ag), copper (Cu), nickel (Ni), palladium (Pd), platinum (Pt), cobalt (Co), rhodium (Rh), iridium (Ir), iron (Fe), ruthenium (Ru), osmium (Os), manganese (Mn), rhenium (Re), scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), zinc (Zn), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), cadmium (Cd), lanthanum (La), lutetium (Lu), hafnium (Hf), tantalum (Ta), tungsten (W), actinium (Ac), lawrencium (Lr), rutherfordium (Rf), dubn
  • Au
  • metal nanoparticles will be bimagnetic and comprise a metal or metal alloy core and a metal shell.
  • Core/shell metal nanoparticles preferably comprise a metal or metal alloy core and a metal shell.
  • Preferred cores are selected from the group consisting of Au, Ag, Cu, Co, Fe, and Pt. Even more preferably, the metal nanoparticles feature a strongly paramagnetic Fe core.
  • Preferred shells are selected from the group consisting of Au, Ag, Cu, Co, Fe, Pt, the metal oxides (e.g., FeO, Fe 3 0 4 , Fe 2 0 3 , Fe x O y (non-stoichiometric iron oxide), CuO, Cu 2 0, NiO, Ag 2 0, Mn 2 0 3 ) thereof, and combinations thereof.
  • Particularly preferred metal core/shell combinations are selected from the group consisting of Fe/Au, Fe(0)/Fe 3 O 4 , and Au/Fe 2 0 3 .
  • a particularly preferred metal nanoparticle is a superparamagnetic Fe/Fe 3 0 4 core shell nanoparticle.
  • the nanoparticles feature an iron(0) core, which is more magnetic than iron oxide, based upon coercivity. This means that smaller nanoparticles can be used (diameter less than about 10 nm), which have the same or greater magneticity than larger iron oxide nanoparticles (diameter of about 200nm).
  • the core of the metal nanoparticle preferably has a diameter of from about 2 nm to about 100 nm, more preferably from about 3 nm to about 18 nm and more preferably from about 5 nm to about 9 nm.
  • the metal shell of the core/shell nanoparticle preferably has a thickness of from about 1 nm to about 10 nm, and more preferably from about 1 nm to about 2 nm.
  • the nanoparticles preferably have a Brunauer-Emmett-Teller (BET) multipoint surface area of from about 20 m 2 /g to about 500 m 2 /g, more preferably from about 50 m 2 /g to about 350 m 2 /g, and even more preferably from about 60 m 2 /g to about 80 m 2 /g.
  • BET Brunauer-Emmett-Teller
  • the nanoparticles preferably have a Barret- Joy ner-Halenda (BJH) adsorption cumulative surface area of pores having a width between 17.000 A and 3000.000 A of from about 20 m 2 /g to about 500 m 2 /g, and more preferably from about 50 m 2 /g to about 150 m 2 /g.
  • BET Brunauer-Emmett-Teller
  • the nanoparticles also preferably have a BJH desorption cumulative surface area of pores having a width between 17.000 A and 3000.000 A of from about 50 m 2 /g to about 500 m 2 /g, and more preferably from about 50 m 2 /g to about 150 m 2 /g.
  • the nanoparticle population is preferably substantially monodisperse, with a very narrow size/mass size distribution. More preferably, the nanoparticle population has a polydispersity index of from about 1.2 to about 1.05. It is particularly preferred that the nanoparticles used in the inventive nanoplatforms are discrete particles. That is, clustering of nanocrystals (i.e., nanocrystalline particles) is preferably avoided.
  • the nanoparticles can be stabilized or non-stabilized.
  • Stabilized nanoparticles preferably comprise an organic monolayer surrounding the nanoparticle core.
  • the term "stabilized” as used herein means the use of a ligand shell or monolayer to coat, protect (e.g., from bio-corrosion), or impart properties (e.g., water solubility) to, the nanoparticle.
  • the monolayer can be comprised of several of the same ligands (i.e., homoligand) or of mixed ligands.
  • Various techniques for attaching ligands to the surface of various nanoparticles are known in the art. For example, nanoparticles may be mixed in a solution containing the ligands to promote the coating of the nanoparticle.
  • coatings may be applied to nanoparticles by exposing the nanoparticles to a vapor phase of the coating material such that the coating attaches to or bonds with the nanoparticle.
  • the ligands attach to the nanoparticle through covalent bonding. The number of ligands required to form a monolayer will be dependent upon the size of the nanoparticle.
  • the ligands comprise functional groups that are attracted to the nanoparticle's metal surface.
  • the ligands comprise at least one group selected from the group consisting of thiols, alcohols, nitro compounds, phosphines, phosphine oxides, resorcinarenes, selenides, phosphinic acids, phosphonicacids, sulfonic acids, sulfonates, carboxylic acids, disulfides, peroxides, amines, nitriles, isonitriles, thionitiles, oxynitriles, oxysilanes, alkanes, alkenes, alkynes, aromatic compounds, and seleno moieties.
  • Preferred organic monolayers are selected from the group consisting of alkanethiolate monolayers, aminoalkylthiolate monolayers, alkylthiolsulfate monolayers, and organic phenols (e.g., dopamine and derivatives thereof).
  • the thickness of the organic monolayer is preferably less than about 10 nm, and more preferably less than about 5 nm.
  • Particularly preferred stabilized nanoparticles are selected from the group consisting of trioctyl-phosphinoxide-stablized nanoparticles, amine-stabilized nanoparticles, carboxylic-acid-stabilized nanoparticles, phosphine-stabilized nanoparticles, thiol-stabilized nanoparticles, aminoalkylthiol-stabilized nanoparticles, and organic phenol-stabilized nanoparticles.
  • the preferred ligands will preferably readily react with the thiol group of the terminal cysteine of the oligopeptide linkage.
  • the nanoparticle surface will preferably be essentially completely covered with ligands. That is, at least about 70%, preferably at least about 90%, and more preferably about 100% of the surface of the nanoparticle will have attached ligands.
  • the number of ligands required to form a monolayer will be dependent upon the size of the nanoparticle (and monolayer), and can be calculated using molecular modeling or ligand modeling methods.
  • nanoparticles may be mixed in a solution containing the ligands to promote the coating of the nanoparticle surface.
  • coatings may be applied to nanoparticles by exposing the nanoparticles to a vapor phase of the coating material such that the coating attaches to or bonds with the nanoparticle.
  • the ligands attach to the nanoparticle through covalent bonding.
  • Nanoparticles can also be non-metal: non-metal oxide (e.g. S1O2), polysilicone, polysilazane, or polysiloxazane, starburst dendrimers, or polymer latex nanoparticles.
  • non-metal oxide e.g. S1O2
  • polysilicone polysilazane
  • polysiloxazane polysiloxazane
  • starburst dendrimers e.g. S1O2
  • PAMAM poly-(amidoamine) dendrimers have defined three-dimensional shape, size, topology, and peripheral functional groups.
  • PAMAM dendrimers are pure macromolecules, with precise molecular weight for each generation (starburst polymers). Dendrimers can be purchased according to desired specifications. Therefore, it is not necessary to synthesize inorganic nanoparticles. The approach is otherwise the same as described herein.
  • One advantage is that the shelf life for dendrimer- based nanosensors is greater than 5 years.
  • PAMAM dendrimer stick to cellulose, and can be printed directly onto paper. This would facilitate mass fabrication of paper microfluidic devices by applying inexpensive desk jet printing technology, as described herein.
  • Chromophore/luminophore particles suitable for use in the inventive assays include any organic or inorganic dyes, fluorophores, phosphophores, light absorbing nanoparticles (e.g., Au, Ag, Pt, Pd), combinations thereof, or the metalated complexes thereof.
  • the chromophore/luminophore particles have a size (maximum surface-to-surface dimension, i.e., diameter) of less than about 100 nm.
  • Suitable organic dyes are selected from the group consisting of coumarins, pyrene, cyanines, benzenes, N-methylcarbazole, erythrosin B, N-acetyl-L-tryptophanamide, 2,5- diphenyloxazole, rubrene, and N-(3-sulfopropyl)acridinium.
  • Specific examples of preferred coumarins include 7-aminocoumarin, 7-dialkylamino coumarin, and coumarin 153.
  • preferred benzenes include l,4-bis(5-phenyloxazol-2-yl)benzene and 1,4-diphenylbenzene.
  • cyanines examples include oxacyanines, thiacyanines, indocyanins, merocyanines, and carbocyanines.
  • Other exemplary cyanines include ECL Plus, ECF, C3-Oxacyanine, C3- Thiacyanine Dye (EtOH), C3 -Thiacyanine Dye (PrOH), C5-Indocyanine, C5-Oxacyanine, C5- Thiacyanine, C7-Indocyanine, C7-Oxacyanine, CypHer5, Dye-33, cyanines (Cy7, Cy7.5, Cy5.0, Cy5.5, Cy3Cy5 ET, Cy3B, Cy3.0, Cy3.5, Cy2), CBQCA, NIRl, NIR2, NIR3, NIR4, NIR820, SNIR1, SNIR2, SNIR4, Merocyanine 540, Pinacyanol-Iodide, l, l-Diethyl-4,4-carbocyan
  • Cyanine dyes are particularly preferred organic dyes for use in the nanoplatforms.
  • the fluorescent cyanine dye is tethered to the nanoparticle and experiences rapid fluorescence quenching by the plasmon of the Fe(0)-core. This is observed as long as the tether is smaller than the Forster-radius of the cyanine dye (5-6 nm for Cy3.0 and Cy3.5, 6-7 nm for Cy5.0 and Cy5.5, and approx. 7 nm for Cy7 and Cy7.5).
  • the maximal length of the tether consisting of the ligand (-2.84 nm) and not more than 12 amino acid residues in the cleavage sequences (up to 4 nm) indicates that shorter cleavage sequences (uPA and MMP' s) are suitable for use with Cy3.x and Cy5.x dyes, whereas the cathepsins are preferably linked to Cy5.x and Cy.7.x dyes to permit optimal quenching of the tethered cyanine dyes.
  • their emission maxima are red-shifted with respect to the autofluorescence of human urine.
  • Multiple cyanines can be linked to a single nanoparticle to create oligoplexing nanoplatforms, to measure the activity of up to four enzymes simultaneously.
  • All four dyes in the UVA or blue region of the electromagnetic spectrum can be excited simultaneously, or each dye can be excited individually.
  • All cyanine dyes have an excitation maximum, which is blueshifted by 20-25 nm with respect to their emission maximum (typical for fluorescent singlet states).
  • Suitable inorganic dyes are selected from the group consisting of metalated and non- metalated porphyrins, phthalocyanines, chlorins (e.g., chlorophyll A and B), and metalated chromophores.
  • Preferred porphyrins are selected from the group consisting of tetra carboxy- phenyl-porphyrin (TCPP) and Zn-TCPP.
  • Preferred metalated chromophores are selected from the group consisting of ruthenium polypyridyl complexes, osmium polypyridyl complexes, rhodium polypyridyl complexes, 3-(l-methylbenzoimidazol-2-yl)-7-(diethylamino)-coumarin complexes of iridium(III), and 3-(benzothiazol-2-yl)-7-(diethylamino)-coumarin complexes with iridium(III).
  • Suitable fluorophores and phosphophores are selected from the group consisting of phosphorescent dyes, fluoresceines, rhodamines (e.g., rhodamine B, rhodamine 6G), and anthracenes (e.g., 9-cyanoanthracene, 9, 10-diphenylanthracene, l-Chloro-9, 10-bis(phenyl- ethynyl)anthracene). 3.
  • fluoresceines e.g., rhodamine B, rhodamine 6G
  • anthracenes e.g., 9-cyanoanthracene, 9, 10-diphenylanthracene, l-Chloro-9, 10-bis(phenyl- ethynyl)anthracene.
  • a quantum dot is a semiconductor composed of atoms from groups II- VI or III-V elements of the periodic table (e.g., CdSe, CdTe, InP).
  • the optical properties of quantum dots can be manipulated by synthesizing a (usually stabilizing) shell.
  • Such quantum dots are known as core-shell quantum dots (e.g., CdSe/ZnS, InP/ZnS, InP/CdSe).
  • Quantum dots of the same material, but with different sizes, can emit light of different colors. Their brightness is attributed to the quantization of energy levels due to confinement of an electron in all three spatial dimensions. In a bulk semiconductor, an electron-hole pair is bound within the Bohr exciton radius, which is characteristic for each type of semiconductor.
  • a quantum dot is smaller than the Bohr exciton radius, which causes the appearance of discrete energy levels.
  • the band gap, ⁇ , between the valance and conduction band of the semiconductor is a function of the nanocrystal's size and shape.
  • Quantum dots feature slightly lower luminescence quantum yields than traditional organic fluorophores but they have much larger absorption cross-sections and very low rates of photobleaching.
  • Molar extinction coefficients of quantum dots are about 10 5 - 10 6 M "1 cm "1 , which is 10-100 times larger than dyes.
  • Core/shell quantum dots have higher band gap shells around their lower band gap cores, which emit light without any absorption by the shell.
  • the shell passivates surface nonradiative emission from the core thereby enhancing the photoluminescence quantum yield and preventing natural degradation.
  • the shell of type I quantum dots e.g. CdSe/ZnS
  • the conduction and valance bands of the shell of type II quantum dots are either both lower or both higher in energy than those of the core.
  • the motions of the electron and the hole are restricted to one dimension.
  • Type II quantum dots behave as indirect semiconductors near band edges and therefore, have an absorption tail into the red and near infrared. Alloyed semiconductor quantum dots (CdSeTe) can also be used, although types I and II are most preferred.
  • These quantum dots can be used to develop near infrared fluorescent probes for in vivo biological assays as they can emit up to 850 nm.
  • quantum dots are selected from the group consisting of CdSe/ZnS core/shell quantum dots, CdTe/CdSe core/shell quantum dots, CdSe/ZnTe core/shell quantum dots, and alloyed semiconductor quantum dots (e.g., CdSeTe).
  • the quantum dots are preferably small enough to be discharged via the renal pathway when used in vivo. More preferably, the quantum dots are less than about 10 nm in diameter, even more preferably from about 2 nm to about 5.5 nm in diameter, and most preferably from about 1.5 nm to about 4.5 nm in diameter.
  • quantum dots can be stabilized or unstabilized as discussed above regarding nanoparticles.
  • Preferred ligands for stabilizing quantum dots are resorcinarenes.
  • Such biological marker-based diagnostics could have numerous applications in precision medicine, particularly for airway diseases, where the availability of point of care devices could lead to the development of personalized treatment strategies for individual patients based on the assessment of the temporal and spatial distributions of inflammatory markers of the airways.
  • the initial diagnostic would involve, in some aspects, sampling fluids from the airways using nasopharyngeal washes (in children), induced sputum, bronchoalveolar lavage, exhaled breath condensates, and the like.
  • Biological samples for other conditions include other biological specimens and fluids, such as blood, urine, saliva, tears, sputum, bronchoalveolar lavage fluid, breath condensate, feces, rectal fluid, vaginal fluid, and the like.
  • a biological sample is collected from a subject and prepared for analysis.
  • the sample can be collected and prepared manually, which includes, for example, manual pipetting of sample, manual protein spot cutting, manual biopsy collection, manual blood draw, and manual microfluidic chip loading.
  • automated process can be used, which includes, for example, use of automated liquid handlers, automated protein spot cutters, automated biopsy collection, automated blood draws, and automated microfluidics chip loading.
  • the biological sample contains secreted proteins, micro-vesicle proteins, exosomes and components thereof, including but not limited to RNA and DNA, enzymes, and the like, which can be used as biological markers indicative of the health status of the subject.
  • the biological sample can be subjected to additional processing for analysis.
  • manual processing approaches can be used including, but is not limited to, manual transfer, manual mixing, and manual phased extraction of samples and chemical components.
  • automated processing approaches can also be used, including, but is not limited to, the use of automated liquid handling devices for sample and chemical component transfer, mixing, and phased extraction.
  • the automated processing step utilizes an automated microfluidics device for sample and chemical component transfer, mixing, and phased extraction.
  • the biological sample will be a liquid biospecimen.
  • the sample is mixed with a pharmaceutically acceptable buffer solution to yield a liquid biospecimen for analysis.
  • the sample (regardless of whether initially in liquid form) is mixed with a solution containing metal cofactors or other components to preserve enzymatic and protein activity in the sample during the processing and analysis.
  • exosome surface recognition elements are used to isolate exosomes from the other biological material sample content.
  • the surface recognition elements include, but are not limited to, exosome specific protein markers including tetraspanins (e.g., CD9, CD37, CD53, CD63, CD81 and CD82), endosome associated proteins (e.g., small Rab family GTPases, annexins and flotillin), proteins involved in exosome biogenesis (e.g., Alix, TsglOl and ESCRT complex), heat shock proteins (Hsp70, Hsp90) and epithelial cell adhesion molecules (EpCam).
  • exosome specific protein markers including tetraspanins (e.g., CD9, CD37, CD53, CD63, CD81 and CD82), endosome associated proteins (e.g., small Rab family GTPases, annexins and flotillin), proteins involved in exosome biogenesis (e.g., Alix, Ts
  • Magnetic capture methodologies are used to capture the exosomes using fixed attachment of the corresponding surface recognition binding sequence.
  • magnetic nanoparticle-based nanosensors i.e., magnetic capture nanosensors
  • the nanosensors (containing bound exosomes) can then be filtered, for example, using magnets to remove the nanosensors from surrounding materials.
  • the exosomes Once the exosomes have been magnetically captured onto a plate, chip, or other collection vehicle, the exosomes are lysed to expose their cargo for further processing, profiling, and target detection.
  • exosome lysis utilizes a plurality of methods such as, but not limited to heat, electromagnetic, acoustic, chemical, photonic, or combination thereof to achieve lysis.
  • isolation and lysing of the exosomes may be carried out on the same microfluidics device, for example, in a microfluidics channel capture region "upstream" of the detection region. It may also be carried out in a separate microfluidics device, before subsequent introduction of the lysed exosomes into the analysis device. Further, conventional isolation and capture techniques can be used before the exosomes are assessing using the technology.
  • surface proteins can be used as biomarkers related to cancer diagnosis. Thus, it is not always necessary to lyse the captured exosomes, as the surface biomarkers themselves can be used for analysis of the sample.
  • nanosensors are used for detection and identification of biological markers in the sample.
  • the nanosensors are designed and/selected based upon the desired target marker selected for detection.
  • the nanosensors can be used to diagnose lower respiratory tract infections by detecting markers associated with infected lower airway epithelial cell infection. These markers include CCL20, TSLP, and CCL3-L1.
  • exosome contents can be used to determine whether the exosomes originated from the upper or lower airway, and further assist in localization of where the infection or other condition may originate from in the subject's body.
  • the nanosensors can also be used for environmental risk assessment in patients with chronic lung disease.
  • the nanosensors can be used for measurement of lower airway remodeling in severe asthma and chronic obstructive lung disease (COPD). Remodeling refers to the process of fibrosis of the respiratory tract, a process linked to progressive decline in lung function in a subset of patients with severe asthma and COPD. There is no treatment or diagnostic currently available. New therapies directed towards epigenetic remodeling are currently being developed. Our method will enable the development and approval of remodeling inhibitors for clinical use by detecting and measuring the presence of fibronectin, IL6, or vimentin, as indicative of progressive airway remodeling.
  • Remodeling refers to the process of fibrosis of the respiratory tract, a process linked to progressive decline in lung function in a subset of patients with severe asthma and COPD.
  • New therapies directed towards epigenetic remodeling are currently being developed. Our method will enable the development and approval of remodeling inhibitors for clinical use by detecting and measuring the presence of fibronectin, IL6, or vimentin, as indicative of progressive airway remodeling.
  • Opportunistic infections are also a significant complication in patients being treated for cancer, immunosuppressed through treatment of rheumatoid arthritis or inflammatory bowel disease, or those with HIV.
  • Pneumocystis pneumonia PCP
  • PCP Pneumocystis pneumonia
  • Invasive aspergillosis is also an opportunistic lung infection occurring in patients undergoing chemotherapy for leukemia.
  • the nanosensors can be used to indicate the presence of distinct host response proteins when the fungus invades the airway in patients immunosuppressed from their leukemia treatment.
  • the nanosensors can also be used for monitoring for transplant rejections or host versus graft disease in lung transplants.
  • Lung transplant patients are treated with intensive immunosuppressive therapy and their physicians treat a fine line between too much immunosuppression (get opportunistic infections), or too little, where they would reject the organ. If a transplant patient could monitor their immune profiles in a real time basis, this would make management of the transplant much easier.
  • the nanosensors can be used for monitoring for response to lung cancer treatment.
  • Mobile biosensing would detect cancer signatures, such as cytokines, proteases, and/or kinases (e.g., MMPs, EMT, etc.), in the airway or circulating in the blood. This would include free proteins and those contained within microparticles (exosomes).
  • embodiments described herein can also be utilized in conjunction with traditional PCR/RT-PCR, real-time quantitative PCR/RT-PCR, and isothermal PCR/RT-PCR methodologies to assess bacterial, viral, and mold infections that are associated with increased or decrease protein level expression.
  • assays according to the invention should not only be considered as a stand-alone technology, but can be used in conjunction with traditional approaches, especially in risk groups that have been pre-identified as being at-risk by genetic testing.
  • the detection involves contacting the prepared sample with the nanosensor (and generally a plurality of nanosensors) to create a reaction mixture.
  • the nanosensors can then be probed or excited using the appropriate energy source.
  • the wavelength used will depend upon the particles used in the nanosensors.
  • the changes in absorption and/or emission of the particles are then detected over a period of time as the target biomarker interacts with the nanosensors (e.g., such as through binding and extension of the recognition sequence).
  • Microfluidic refers to techniques manipulating, controlling, and/or analyzing small volumes of (fluid) samples, generally ranging from microliters (10 "6 ) to picoliters (10 "12 ).
  • microfluidic technology is employed to introduce rapid, high-throughput sample collection, sample processing, sample profiling and target detection.
  • the microfluidic device will interface with a mobile computing device such as, but not limited to, a smart phone or smart tablet. Mobile computing devices including tablets, smart phones, hand held computers, laptops, etc.
  • a stand-alone, battery- powered diagnostic platform with rapid analysis times and multiplexing capabilities will achieve statistical significance of measured quantities, with minimum consumption of human fluids ( ⁇ 5 ⁇ 1 per sampling) using a hand-held smartphone-based system that can be used as a diagnostic tool by patients and medical professionals.
  • the microfluidic device utilizes a plurality of detection sites in fluid communication with a micro-scale sample inlet well for introducing and analyzing a small volume of sample fluid through capillary force.
  • the microfluidic device includes a substrate having a first major surface (e.g., top surface) and an opposing second major surface (e.g., bottom surface).
  • the top surface comprises a sample application region and at least one detection region.
  • the detection region may generally occupy a space in the substrate having a volume of about 100 pL to about 1 ⁇ .
  • the sample application region is configured to receive an amount of the test sample (e.g., from about 10 ⁇ _, to about 5 mL).
  • the detection region comprises at least one nanosensor immobilized therein/thereon for detection of a biomarker in the sample.
  • the substrate can be a woven or nonwoven solid support, which can be elongated or of various other geometric configurations.
  • the sample application region is in capillary flow communication with the detection region whereby a sample absorbed on the microfluidic substrate may flow by capillary action through the solid support from the sample application region to the detection region. Accordingly, there is a region of the substrate between the sample application region and the first detection region that defines a first path of flow of the material (i.e., a channel in/on the substrate from the application region to the first detection region).
  • the substrate can be a test strip or ribbon (with two terminal ends) or other suitable geometric shape to facilitate flow of the material from the sample application region to the detection region.
  • the top surface of the substrate will typically be substantially planar or flat, so as not to impede the flow or the sample, and comprise a porous membrane matrix with a suitable thickness that allows flow through of the test sample (liquid).
  • the substrate can include a plurality of detection regions, each in fluid communication with the sample application region. This can include a plurality of channels extending between the sample application region and a respective detection region. It can also include a plurality of detection regions linearly or sequentially spaced along a single channel.
  • any suitable approach can be used to create hydrophobic regions in the substrate and thus define the hydrophilic channels to direct the flow of the test sample from the sample application region to (and past) the detection region(s).
  • the configuration of the channel(s) can be created using photosensitized polydimethylsiloxane (PDMS).
  • PDMS photosensitized polydimethylsiloxane
  • the porous substrate is soaked in the PDMS and then the PDMS-soaked matrix is be patterned using a mask and an appropriate light source shone through the mask to transfer the mask pattern to the PDMS-soaked porous substrate.
  • the PDMS in the exposed regions is cross-linked and the PDMS in the unexposed regions can be rinsed away using an organic liquid. These rinsed regions are hydrophilic and define the channels through which the sample flows.
  • the photosensitized PDMS is amenable to large-scale mass production of sensor chips at ultra-low cost. Wax printing can also be used to pattern and define the channel architecture for higher
  • the developed nanosensors are pre-immobilized in each detection region of the substrate.
  • An example of an approach for immobilization on cellulose is illustrated in Fig. 6.
  • the nanosensors contain different recognition sequences for the target analyte or biomarker in the sample, and can be selectively pre-immobilized in each detection region, for example, through a needle-based pressure pull of reagents at the edge of the region.
  • the detection regions containing the nanosensors are spatially separated. Therefore, a bandpass filter and a mask featuring pinholes are sufficient to observe the presence or absence of the detectable signal from clearly isolated regions.
  • the collected sample is added to the sample application region and flows by capillary action to (and over/through) each detection region containing the nanosensors.
  • the microfluidic substrate can be any material that facilitates flow of the biological sample from the sample application region to the detection region(s) via capillary action.
  • any porous or sorbent material (or combination of sorbent materials) can be used for the substrate, as long as it is capable of absorbing, either by capillary action or otherwise, molecules that pass through/along the substrate.
  • various sorbent materials (or combinations of sorbent materials) can be used, such as 100% cotton cellulose fiber (e.g., Whatman #1 filter paper or a similar type of material). Cellulose-based materials are particularly suited for use as test strips.
  • the device further comprises a wicking pad at or near the terminal end of the substrate (opposite the sample application region), with the detection region(s) being positioned therebetween.
  • the sample after being applied to the sample application region, wicks along the channel(s) in the substrate, causing the fluid sample to pass over the detection region(s) as it is "pulled' towards the wicking pad positioned near the terminal end of each channel (opposite the sample application region/inlet).
  • the detection region is positioned nearer to the sample application region than to the wicking pad.
  • suitable materials for the substrate include other lateral flow assay materials, such as nylon, paper, polymers (e.g., polyester, polystyrene, polyacrylamide, nitrocellulose), and activated forms thereof.
  • a miniature foil heater/thermocouple can be integrated into the substrate at the detection region, if desired, to facilitate interaction of the sample with the nanosensors.
  • the test article further includes a cartridge for containing the solid support.
  • the heater can also be part of a cartridge for the solid support, so long as it remains in physical contact with the solid support.
  • Both the cartridge and solid support can be single use and disposable.
  • the cartridge may also be reusable.
  • the cartridge can be made from bio-degradable plastic.
  • the cartridge body will generally include a bottom support structure configured to be positioned underneath the solid support (and adjacent the second major surface), and a top support structure configured to be position on top of the solid support (and adjacent the first major surface).
  • the top support structure includes openings for depositing the biological sample and viewing changes in the nanosensors in detection region(s).
  • the opening for deposition of the biological sample defines a sample inlet well with a sidewall extending from the opening to a bottom wall for containing the sample, where the bottom wall of the microwell is defined by the top surface of the solid support.
  • the volume defined by the inlet well can range from about 10 ⁇ , to about 5 mL.
  • the opening above the detection microwells can be a true "open" through- hole, or may simply be a clear viewing window.
  • the device further includes a housing that can be attached to a smart device and analysis cartridge.
  • a permanent (non-disposable) housing can be used, which contains the detection optics and electronics necessary to control device heating and the assay timing.
  • the cartridge contains the sample well, microfluidic substrate, conductivity pads, and a thin film foil heater.
  • a sample e.g., ⁇ 2mL of BALF fluid or ⁇ 2mL of (lysis) buffers containing biospecimens
  • a parallel fluidic manifold as depicted.
  • the substrate includes 8 parallel channels with 6 of the channels containing a single optical-based nanobiosensor (OBN) readout pad.
  • OBN optical-based nanobiosensor
  • the 7th channel contains a positive control, and the 8th channel a negative control.
  • the biosensors will be attached to the substrate using, for example, polysilazane attachment chemistry. All types of described nanosensors, including cytokine sensors described herein, as well as protease sensors, and arginase sensors, are adaptable to the microfluidics device.
  • the polysilazane chemistry also allows a new approach to the nanosensor development where the primary florescent indicator is embedded directly into the polysilazane matrix as it is applied and the probe and quencher are attached afterward to the surface of the matrix. It will be appreciated that a variety of alternative cellulose linking chemistries are known in the art for immobilizing the nanosensors in the detection region(s).
  • the nanosensor detection regions are positioned near the sample application region/inlet to reduce potential protein adsorption to the substrate fibers.
  • the substrate can be passivated/blocked using BSA or other blocking proteins (e.g. casein), or the substrate could potentially be coated with poly(ethylene oxide) or poly(ethyleneglycol) to reduce peptide/protein absorption.
  • a wicking material is positioned at the terminal end of each channel to facilitate capillary flow of the entire volume of the sample across each detection zone.
  • Readout time can be automated.
  • a conductivity detector can be used to mark the 0 time point when the sample inlet is wetted with the sample to quantitate the flow rate of the sample (i.e., the volume of sample that has passed over the detection region over a certain period of time).
  • the readout/detecting can be carried out anywhere from about 1 minute to about 20 minutes after the sample is introduced into the device. If sample viscosity varies significantly and adversely affects the rate of flow in the substrate, additional conductivity detectors can be spaced along the channel so that the readout time is based upon the total volume of sample passed over the nanosensor detection region(s). Fluorescence signal is best detected from the top 10 ⁇ of the substrate so flow can be optimized to allow diffusion through the matrix to maximize signal. Channel dimensions and substrate wettability can be adjusted to provide a readout time of ⁇ 10 min.
  • a change may be detectable in the detection region.
  • the fluorophore or quencher
  • the fluorophore is released from the nanosensor such that the fluorophore is no longer quenched.
  • the fluorescence can be detected in the detection region, indicating that the target protease is active and present in the sample.
  • the maximum absorption of the TCPP is near 420 nm, so an inexpensive 420nm LED or any other time of laser diode can be used for excitation of nanosensors using TCPP as the detectable particle.
  • Collimating and beam shaping optics can also be used to create a narrow excitation line across the channels where the detection spots were laid down.
  • COMSOL multiphysics simulations can be used to adjust the beam shaping optics to provide even illumination across all of the detection.
  • the fluorescence emission will be collected through a 660df 20nm bandpass filter and imaged on a cell phone camera CCD for detection.
  • the nanosensors are not limited to the TCPP-cyanine FRET pair exemplified in the examples. Any FRET sensing pair can be used. If other FRET pairs are used then the excitation wavelength and optical filters can be modified accordingly.
  • the turnover rates for the analyte enzymes are highest at 36°C so a miniature foil heater/thermocouple can be integrated into the substrate.
  • the heating can be controlled with PID software.
  • the foil heater and thermocouple can also be moved off the disposable cassette and into the permanent housing with the optical detection system.
  • the extent of the heated zone on assay performance can be identified in terms of potential diffusion and evaporation issues. For example, if a 500 ⁇ L ⁇ sample of BALF fluid is divided into 8 channels (6 for analytes and 2 for controls) that means each sensor will be exposed to about 60 ⁇ ⁇ of fluid. Clinically relevant biomarker concentrations are expected to be 10 "11 to 10 "8 M.
  • the total number of moles for the lower expected concentrations should therefore be ⁇ 6xl0 "16 moles which is well above the detection limit of the sensors. It will be appreciated that larger BALF volumes can be collected and so the volume used can be increased or the nanosensors can all be deposited in a single channel to further increase the number of moles of analyte they are exposed to. In this approach, the nanosensors would utilize a variety of different fluorophores with minimal spectral interference. Or, alternatively, the density of the sensors or the sensing region could be increased.
  • the device will simultaneously measure at least 1 and as many as 20 biomarkers. Preferably, the device will measure between 3 and 15 biomarkers. More preferably, the device will simultaneously measure between 6 and 10 biomarkers.
  • sample processing can be added to the sample inlet to filter out particulate matter so that centrifugal pretreatment of the sample will not be necessary.
  • the most likely material for such pretreatment will be a polysulfone filter. This step will result in further reduced costs for the whole measurement process.
  • the relative optical density image analysis will be accomplished via a cross-platform smart device application written in Java for quantitative fluorescence measurement.
  • a collimating lens and a band-pass center wavelength of 1 650 nm (625 nm - 675 nm) will be attached to a 5xlens for eliminating unwanted background noise and enhancing the signal-to-noise ratio for imaging of TCPP- fluorescence.
  • diagnostic results are displayed on smart phone of smart tablet and can be used to assist disease management and progression.
  • Numerous learning algorithms are capable of capturing significant correlations between data sets by modeling the distribution of the data by introducing latent, or hidden, variables. Typical examples for this methodology are neural networks or factor analysis.
  • the major advantage of Generative Topographic Mapping Algorithms (GTM) is that they can be extended to allow non- linear transformations while remaining computationally tractable. See Fig. 8.
  • GTM is based on a constrained mixture of Gaussian functions whose parameters can be optimized using EM (expectation-maximization) algorithms.
  • GTM algorithms are principally superior to Self- Organizing Map (SOM) algorithms, which do not define probability densities.
  • GTM algorithms can be successfully utilized for the visualization and fast analysis of large arrays of chemical data.
  • Various data distribution functions resulting in various probability distribution functions e.g. data density and property landscapes
  • GTM algorithms are very suitable for processing BIG DATA, if the necessity should arise in the future.
  • the smart device-based detection technology can be used for detection of protein signatures indicating the presence of regional epithelial injury or infection in airway samples or exhaled breath condensates; a nanosensor for point of care testing for monitoring the airway for the presence of inflammation from infection and/or environmental injury; a point of care device to diagnose or to triage children or adults with asthma for the presence of lower respiratory tract involvement; a smart-phone based device for real time monitoring of the impact of environmental exposures in home or industrial environments on the health of airway; a device for assessing progression of infection or resolution of infection in an outpatient setting; and a device for measuring response to therapy in patients with asthma or viral induced airway infections.
  • the use of the present nanosensors enables limits of detection (LOD) for proteases, post- translational modification enzymes (e.g., Arginase) and Cytokines/Chemokines that are significantly lower than picomolar.
  • LOD limits of detection
  • This technique is approaching femtomolar and sub- femtomolar LOD for numerous proteases and arginase.
  • cytokines the technology is currently at LODs of about 10 "14 moles L "1 .
  • immunoassays which rely on antibodies or antibody fragments, have LOD that are only sub-picomolar (10 "12 to 10 "13 moles L "1 ).
  • the present nanosensors can achieve LODs of from about 10 "9 M to about 10 "18 M.
  • microfluidic devices have the advantage of processing the liquid biopsies within minutes, whereas competing technologies require several hours to reach their (respective) maximal LOD. Furthermore, microfluidic devices are capable of lowering the LOD by another one or two orders of magnitude. Therefore, the combined microfluidics and nanosensor technology is significantly faster and more sensitive than competing technologies.
  • Microfluidic technology also facilitates multiplexing.
  • biomarkers e.g., several proteases, cytokines and kinases
  • the technology permits simultaneous detection of different classes of biomarkers in a single platform— an approach previously limited to mass spectrometry.
  • the level of sensitivity far outperforms what can be achieved with MS, and also permits real-time detection and results. This enables the "Barcode Detection Principle", which is looking at 10-20 biomarkers for each disease. Looking at multiple biomarkers permits the differentiation between related diseases (e.g., lung inflammatory diseases vs. asthma) and the detection of diseases in very early states.
  • the nanosensor and methods measure the activity of target proteins and enzymes, as well as their concentrations and relative ratios. This is unique, because all antibody- based detection technologies utilize epitopes for target binding. They are not capable of sensing whether the enzymes are active or not. These enzymes are usually expressed as zymogens (inactive enzymes), which require either proteolytic activation or activation via posttranslational modification. Many of them show signaling activity when they are zymogens and enzymatic activity after activation. For the diagnosis of a disease, it is very important in what state (zymogene vs. active enzyme) a biomarker is. The present technology can provide that insight.
  • this detection technology can be extended to capturing exosomes and measuring the activity of enzymes that are either bound to the exosomes surfaces or (after lysing) incorporated into exosomes. Further, lysis of the exosomes will permit the determination of the activity of enzymes or the concentration of cytokines that were contained in the exosomes' interior. Lysed exosomes can also be assessed for RNA and metabolites as markers of exosomal activities and function, as well as inflammation-induced changes in protease, kinase, or cytokine activity.
  • Cancer-induced changes in phosphatases e.g., alkaline phosphatase
  • DNA/RNA synthesizing/modifying enzymes e.g., ribonucleotide reductases or DNA/RNA helicases
  • the detection technology also permits direct or indirect assessment of exosomal stability and activity.
  • exosomes can alternatively be prepared and extracted using conventional technique of centrifuging the samples at low to high g for optimum duration, yielding a pellet containing exosomes that can be characterized using nanosensors described herein, in order to monitor and characterize exosomal activities and function defined by quantification of changes in protease, cytokine, and/or kinase secretion.
  • the phrase "and/or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed.
  • the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
  • the present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).
  • a nanosensor for detecting cytokine activity was prepared.
  • the oligopeptides are synthesized by solid phase synthesis.
  • the fluorescent dye is attached while the oligopeptide is still on the column.
  • the oligopeptide is dialyzed, lyophilized and then purified by means of gel chromatography. Typical purities are 85% after synthesis, 95% after dialysis and 99++ percent after gel chromatography.
  • the following supramolecular recognition sequence was used for IL-6: RPAQAWMLG (SEQ ID NO:21)
  • a commercially available dry resin (purchased from Peptides International) with the starting amino acid (500 mg) was placed in the reaction vessel.
  • 4 ml DCM was added until all resin beads are immersed to swell the resin in the DCM for 20 minutes. This helps the resin to react well with the next amino acid to be added.
  • the resin suspension was gently swirled for 20 min. Then the DCM was removed by filtration under vacuum.
  • the first N a Fmoc deprotection was performed using 4 ml 80/20 DMF/diethylamine solution. The mixture was stirred for 1 min and the solution was then removed by vacuum filtration. The same step was repeated within 10 min. The resin was then washed five times with 4 ml of DMF by stirring it for 30 s. The solvent was removed by vacuum filtration.
  • the desired peptide sequence was assembled in a linear fashion from the C-terminus to the N-terminus (the C_N strategy) by repetitive cycles of N" deprotection and amino acid coupling reactions. Deprotection and coupling processes are repeated until the desired sequence is obtained.
  • the TCPP tetrakis-4-carboxyphenyl-porphyrin
  • the TCPP tetrakis-4-carboxyphenyl-porphyrin
  • Resin:TCPP molar ratio was 1 :3. After the addition of dye to the resin, it was swirled for 24 hrs at RT and the excess dye was washed off with DMF solution.
  • cleavage cocktail trifluoroacetic acid/water/triisopropyl silane 3.8/0.1/0.1
  • the resin was swirled gently for 3 hrs at RT. Then it was filtered into 20 ml cold diethyl ether. The peptide sequence was precipitated and was washed with cold ether three times. The precipitate was isolated by centrifugation (10000 rpm, for 5 min). Finally, argon was applied to dry the product for safe storage.
  • the oligopeptide was purified by quantitative HPLC.
  • MALDI-TOF Microx-assisted laser desorption/ionization-time of flight mass spectrometry
  • the nanosensors can be calibrated according to the following procedures. Calibration of the IL-6 nanosensor was conducted with commercially available recombinant human IL-6 to validate the sensing capabilities. The calibration curves are recorded as a function of IL-6 concentration. Commercially available recombinant human IL-6 purchased from BD Biosciences was used. Concentrations ranging from 1.0 x 10 "16 mol dm "3 to 1.0 x 10 "8 mol dm “3 were utilized for the calibration process. Plate reader technology was used to measure the fluorescence signals. Five replicates from each concentration were analyzed in order to get accurate data. Recombinant human IL-6 is stored at -80°C. The assay procedure was as follows:
  • a Biotek FL800 plate reader (tungsten halogen lamp, excitation bandpass filter: 421 ⁇ 10 nm, analysis bandpass filter: 650 ⁇ 25 nm) with 96-well plates was used. The plate reader was set to 25°C. Distilled water was used for all procedures.
  • the supramolecular recognition sequences were developed according to the following procedure: A) The primary structure of the target was retrieved from a data bank (e.g. UniProtKB or Protein Data Bank). B) The tertiary structure of the target was determined and then refined utilizing protein structure predicting software, such as the GalaxyWEB protein structure prediction cluster, developed by Dr. Seok at the Computational Biology Lab, Seoul National University, Korea. C) The primary structure of a monoclonal antibody (MAB) fragment against the target was retrieved from a data source (e.g., Protein Data Bank, SciFinder, or information provided by the vendors of antibodies). A tertiary structure was generated using GalaxyWEB.
  • a data bank e.g. UniProtKB or Protein Data Bank
  • MAB monoclonal antibody
  • a docking procedure between the target and antibody chain is simulated using the Mobyle platform developed by the Institut Pasteur Biology IT Center, Paris, France. This procedure reveals the "true” epitope of the target.
  • a final “site finder” procedure is then performed on the Mobyle platform docking the epitope of the target to the improved structure of the antibody (Ab) fragment. This structure reveals the peptide sequence of the Ab fragment that is actually responsible for binding to the target.
  • this sequence was synthesized and used as a recognition sequence for the nanosensors. If it was not linear, or if the LOD of the resulting nanobiosensor was not at least 10 "14 M, the in silico procedure was be repeated using the structural information from another mAb.
  • EBC samples from the UTMB biorepository bank were analyzed using nanosensors containing detection sensors for various biological markers.
  • the EBC samples had been collected from three human subjects who did not show any signs of asthma, and three who were diagnosed with mild asthma (no corticosteroids).
  • ADAM 33 disintegrin and metalloproteinase domain-containing protein 33
  • MMP's 8 and 9 neutrophil elastase
  • NE neutrophil elastase
  • MCP-1 monocyte chemotactic protein 1
  • MIP-1 macrophage inflammatory protein 1
  • mice were treated with phosphate buffered saline (PBS, group A), the immunostimulant polyinosinic:polycytidylic acid (poly IC) was administered to eight mice (group B) to induce lung injury and inflammation, and five mice were given Respiratory Syncytial Virus (RSV) to induce airway infection (group C).
  • PBS phosphate buffered saline
  • poly IC immunostimulant polyinosinic:polycytidylic acid
  • RSV Respiratory Syncytial Virus
  • BALF Bronchoalveolar lavage fluid
  • MMP-8 neutrophil elastase
  • NE neutrophil elastase
  • MIP-1 granzyme B and MIP-1
  • Each of the nanoplatforms bearing CD9, CD63 and CD81 has different binding efficacies, or, alternatively, the three tetraspanins are not available in equal concentrations at the exosomes' surfaces.
  • exosomes Incubation of at least lh is recommended if the exosomes should be magnetically captured for subsequent lysis and analysis of their protein, DNA and RNA content. However, this is not necessary for detecting the presence of exosomes.
  • a new design paradigm for a dielectric actuator for fabrication of both valves and pumps on microfluidic devices is described.
  • An exemplary design is shown in Fig. 14.
  • the fluidic channel is sandwiched between a liquid compliant electrode (top) and a planar electrode (bottom).
  • the dielectric layer and fluidic channel between them changes shape due to the force generated between the electrodes.
  • the dielectric is not compressible, the dielectric layer must change shape as discussed above.
  • the easiest method to relieve the stress is to compress the fluidic channel thus pinching off the channel between the 2 electrodes. This effectively forms a valve, which under unpowered conditions is normally open.
  • a thin insulating (dielectric) layer may be applied between the bottom electrode and the fluidic channel.
  • This layer may formed of silicones, titanium oxides, titanates (e.g., Sr and Ba) or perovskite materials that have significantly higher electrical breakdown potentials than PDMS or other types of elastomers.
  • such surfaces have functionalities that make them suitable for modification and the attachment of sensing species as described below.
  • a peristaltic pump By creating 3 normally open valves along a channel (Fig. 15) a peristaltic pump can be created that is improved over pneumonic actuation. That is, pneumatic actuation requires significant off chip equipment, and the solenoid valves used to operate the gas lines are power intensive. In addition, liquid/air tight seals with the chip must be made for all of the valve control lines. Dielectric valves, conversely, do not require air/liquid tight interfaces. Connections to electrical power supplies can be made using small, flexible wires or pins. The capacitance of the actuators is low and so little power is required to drive the valves. This allows high voltage/low current power supplies to be used for actuation. Such supplies could even be powered using computer USB ports. In such designs, a single power supply can be used with several high voltage switches. The switches can be opto-diodes. Such switches can work at speeds of over a kHz. They are compact and inexpensive.
  • FIG. 16 A design for an exemplary robust point of care (POC) system that can be used for disease diagnosis is shown in Fig. 16.
  • POC point of care
  • two recirculating microfluidic loops are used to first concentrate a biologically significant molecule or species (e.g., exosomes), from a biological sample (e.g. blood, sputum, nasal lavage, etc.) and then to detect the biological marker(s) using a variety of sensing platforms.
  • a volume of several mL of biological fluid can be loaded into the loop displacing a sterile PBS type buffering system.
  • the fluid is first loaded into an input reservoir. Valves VI and V3 are opened while valves V2, V4, and V5 are closed.
  • the pump in loop one (LOOP1) is opened and the sample drawn into the loop as the PBS is displaced and sent to waste. Once LOOP1 is filled. Valves VI and V3 are closed and valve V2 is opened. The sample is then recirculated over the capture pad CP1 until a sufficient quantity of the analyte (e.g., species, exosome, etc.) is captured. The capture may be attained using a variety of affinity agents including but not limited to antibodies, aptamers, peptide aptamers, Fab fragments, etc. Once captured and concentrated on CP1, pump PI will be stopped. Valves V4, V5, V7, and V8 will be opened. Pump PI valves will be closed along with valve V6.
  • analyte e.g., species, exosome, etc.
  • the device material used to fabricate the microfluidic channels and the compliant electrode can be a silicone, modified silicone or acrylic elastomer. These materials are highly transparent.
  • the bottom electrode can be made from indium tin oxide (ITO) deposited on a thin glass slide, as the ITO and slide are transparent and will allow easy observation and troubleshooting of the device.
  • ITO indium tin oxide
  • the non-compliant electrode material can be any electrically conducting substance.
  • a high dielectric coating will preferably be used. This coating may be a silicone, acrylic, cellulose acetate, or barium (or strontium) titanate, among others.
  • the bottom non-compliant electrode may cover the entire bottom plane of the device and serve as a ground plane or high voltage plane.
  • valves would then be actuated by applying a potential to the compliant electrodes or grounding them if they are otherwise floating.
  • all of the compliant electrode channels can be grounded and the non-compliant electrodes patterned individually on the glass slide.
  • the valves could be individually actuated either through switching the ground electrodes from floating to grounded or be applying a potential to one of more of the patterned non-compliant electrodes.
  • a fluorescent dye preferentially with a high fluorescence or phosphorescence quantum yield (e.g. Rhodamine B) will be dissolved in an inorganic solvent (e.g. a hydrocarbon, an ether, or an ester), together with a polysilazane designer polymer:
  • a mixture of rhodamine B, a polysilazane designer polymer (x, and z can be varied from 0- 100 percent, y from 0-25 percent); n,m are between 1 and 100,000 will be dissolved in a solvent without functional groups featuring polar hydrogens (-COOH, -OH, - H, -SH, HX, etc).
  • n,m are between 1 and 100,000 will be dissolved in a solvent without functional groups featuring polar hydrogens (-COOH, -OH, - H, -SH, HX, etc).
  • 1-10% by weight of the polysilazane designer polymer and 0-5 percent by weight of the polyethylene glycol derivatives are dissolved, leading to a stable "ink” (store in the dark and protect from humidity).
  • This ink can be used to print spots of immobilized dyes onto cellulose paper.
  • the surface will be hydrophilic, so that aqueous buffers can react with it.
  • An exemplary protocol is below.
  • step B Printing of the Designer-polysilazane supported fluorescent dye (every fluorescent dye possessing a functional groups featuring at least one polar hydrogen) onto the paper.
  • the polar hydroxy groups react either with the Si-H groups of the designer polysilazane under formation of H 2 and O-Si bonds, or they exchange Si-NH- against Si-O-, and NH 3 is eventually released.
  • step B follows. This technology is forming sub-micrometer layers on immobilized fluorescent dyes on paper (or, more generally, substrates).
  • B l Printing of an oligopeptide used in posttranslational sensors featuring 1-5 serines at their C-terminal ends (e.g., GRRRRRRRGSSS, SEQ ID NO:76), dissolved in DMF or any other dipolar aprotic solvent (DMA, acetonitrile) or methyl-group capped oligoethylene glycol, in which the oligopeptides are soluble.
  • DMA dipolar aprotic solvent
  • methyl-group capped oligoethylene glycol in which the oligopeptides are soluble.
  • the -OH groups of the serine units at the C- terminal end react with the designer polysilazane under formation of H2 and O-Si bonds, or they exchange Si-NH- against Si-O-, and H3 is eventually released.
  • a FRET partner or quencher attached, which is matched with the emission spectrum of the immobilized dye.
  • B2 The same reaction as described under B l can be applied to print the "classic" consensus sequences onto the very thin layer of immobilized fluorescent dye. Again, we add 1-5 serines to the C-terminal end of the polymer. Principally, threonine or tyrosine would work as well.
  • the first oligopeptide that is printed onto the thin layer of designer-polysilazane immobilized fluorescent dye contains the paratope for binding the target (e.g., cytokines, growth factors, enzymes that don't cleave/chemically modify the oligopeptide tether). It contains 1-5 serines at its C-terminal end, as previously described. Serines are optimal, because they are hydrophilic and facilitate the aqueous buffers containing the target proteins to flow over the nanosensor capture area (instead of around it).
  • the second oligopeptide contains the epitope of the original target.
  • C2 Classic protease-cleavage reaction, except that the quencher is removed.
  • C3 The real target displaces oligopeptide 2, and together with it the quencher.
  • Oligopeptide 1 to be bound to the surface of the polysilazane designer polymer: A V Y YC QQNNEDPRTF GGGTK S S S G (SEQ ID NO:77).
  • Oligopeptides 2 Dye-QFVKDLLLHLKKLFREGRFNG (SEQ ID NO:78), Dye- QFVKDSLLHLKKLFREGRFNG (SEQ ID NO:79), Dye-QFVKDSLLHLSKLFREGRFNG (SEQ ID NO:80), and Dye-QFVKDSLLHLSKLFRESRFNG (SEQ ID NO:81).
  • One, two and three serines are introduced into the developed sequence. Each serine weakens the binding constant by approximately one order of magnitude.
  • any natural or unnatural amino acid, as well as D-amino acid at any position in the sequence can be used to weaken the binding and thus provide a greater range of measurements (if mixtures of oligopeptides 2 are bound to oligopeptide 1).
  • the invention is useful in the assessment of inflammatory response in the airways induced by the exposure of the airway to pathogens, allergens, and toxins by characterizing and quantifying the response of a patient' s lower respiratory tract based on the analysis of protein patterns in the airway fluids and utilizing a novel sample collection device interfaced with smartphone and mobile computing platform.
  • proteins can be free-floating or bound in nanovesicles (exosomes).
  • RSV infections produce a variety of clinical syndromes including upper respiratory tract infections (with or without recurrent otitis media) and lower respiratory tract infections (LRTIs). Although the majority of infections produce an uncomplicated URI, in -2% of predisposed children RSV can spread into the lower airways, producing LRTI. RSV is consequently the most common cause of childhood LRTIs, responsible for 120,000 hospitalizations for LRTI (bronchiolitis) in the US annually, and represents the leading cause of infant viral death worldwide. Apart from its acute morbidity, severe LRTIs are associated with reshaping the pulmonary immune response, producing Th2 polarization and enhancing susceptibility to recurrent virus-induced wheezing through the next several decades of life. The mechanisms by which LRTIs reprogram the pulmonary immune system are not fully understood.
  • LRTIs from RSV are due, in part, to secreted signals from lower airway cells that modify immune response and trigger airway remodeling.
  • hBECs trachea
  • hSAECs small airway bronchiolar cells
  • CCL20/MIP3a was the most active mucin-inducing factor in the RSV-infected hSAEC secretome, and was differentially expressed in smaller airways in a mouse model of RSV infection.
  • hBECs and hSAECs were established by transducing primary cells with human telomerase and cyclin- dependent kinase (CDK)-4 retrovirus constructs (22, 23).
  • hBECs and hSAECs were grown in basal medium supplemented with growth factors (Lonza, Walkersville) in 10 cm Petri dishes in a humidified incubator with 95% air/5% CO2 at 37 °C. At 80-90%> confluence, the medium was changed, fresh basal medium without growth supplements was added to the plates, and the cells infected with pRSV (MOI 1.0) for 24h.
  • pRSV MOI 1.0
  • CM Conditioned Medium
  • CM primary human bronchial epithelial cells
  • phBECs primary human bronchial epithelial cells
  • phSAECs phSAECs
  • CM for UV-inactivated RSV- infected cells was used to stimulate hBECs at a 1-25% (vol/vol) concentration for the indicated times. UV inactivation was as previously described (24).
  • 20 ⁇ . of RSV-CM was mixed with anti-CCL20 Ab (R&D Systems, Minneapolis, MN).
  • Exosome preparation Exosome isolation was performed by differential centrifugation at +4 °C to minimize protein degradation. Cells were removed by low-speed centrifugation at 400 x g, 10 min. The cleared supernatant was then sequentially centrifuged at 2000 x g for 15 min and 10,000 x g for 30 min to remove any remaining cell debris/microvesicles. Exosomes were finally pelleted by ultracentrifugation at 100,000 x g for 2 h and washed in PBS (without Ca++ or Mg++) at 100,000 x g, 60 min. After washing, the pellet was resuspended in a total of 200 ⁇ _, of PBS.
  • Exosome size was estimated by dynamic light scattering using a Malvern High-Performance Particle Sizer (Malvern Instruments, Westborough MA). Data acquisition and analysis were performed using Dispersion Technology Software (DTS, V4.1.26.0) configured for HPPS analysis. Each experimental group had three independent replicates.
  • DTS Dispersion Technology Software
  • sucrose cushion-purified RSV The human RS V A2 strain was grown in Hep-2 cells and prepared by sucrose cushion centrifugation as described. The viral titer was determined by a methylcellulose plaque assay. pRSV aliquots were quick-frozen in dry ice-ethanol, and stored at -70 °C until use.
  • IF Immunofluorescence microscopy. Airway cells were plated on cover glasses pretreated with rat tail collagen (Roche Applied Sciences). The cells were fixed with 4 % paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, blocked in 10% goat serum, and incubated with primary rabbit polyclonal Ab to cytokeratin-7 or 19 as indicated. After incubation with Alexa-goat anti-Rb Ab, cells were washed and counterstained with 4', 6- diamidino-2-phenylindole (DAPI). The cells were visualized with a Zeiss fluorescence LSM510 confocal microscope at 63x magnification. IF of paraffin-embedded mouse lung was performed following standard protocols.
  • lung sections (5- ⁇ ) were deparaffinized in xylene and hydrated in ethanol (100%) - 70%). Sections were washed with deionized water. Antigen unmasking was done with 1.0 mM EDTA, pH8.0. Sections were washed in H2O and blocked with 10%> goat serum for 1 h at room temperature in the dark, followed by primary antibody against CCL20 (1 :200 dilution, Abeam) overnight at 4 °C in the dark. Sections were washed with TBS-Tween (0.1%>) buffer and incubated with goat secondary Ab conjugated with Alexa 568 (DAKO) for lh at room temperature in the dark. Sections were washed in TBS-Tween (0.1%), incubated with DAPI for 1 min, mounted on coverslips and visualized by confocal microscopy (Zeiss) and photographed at 63x magnification.
  • DAPI Alexa 568
  • Apoptosis assay Apoptosis was measured using a commercial annexin V-FITC apoptosis detection kit following the manufacturer' s protocol (BioVision, Milpitas, CA). Briefly, hBEC and hSAEC cells (lxlO 6 ) were infected with or without RSV (MOI 1.0) for 24h. Cells were dislodged with Accutase (Millipore), washed once with PBS and incubated with 5 ⁇ _, of annexin V-FITC and 5 ⁇ _, of Propidium Iodide (PI) in 500 ⁇ _, of binding buffer for 5 min at room temperature in the dark. Annexin V-FITC and PI labeling was measured by flow cytometry.
  • TEM Transmission Electron Microscopy
  • a 10 ⁇ aliquot from the exosome suspension was diluted in deionized water, applied to 200 mesh Formvar/carbon coated copper grids (Electron Microscopy Sciences) for 10 min at room temperature (24 °C) and negatively stained with 2% uranyl acetate (UA).
  • the grids were examined in a Philips CM- 100 transmission electron microscope at 60 kV FEI (Thermo-Fischer). Exosome images were acquired with a Gatan Orius 2001 charge-coupled device (CCD) camera.
  • CCD charge-coupled device
  • Proteins were digested with 1.0 ⁇ g LysC-tr (Promega) for 12 h at 37 °C and then diluted 4: 1 with 50 mM ammonium bicarbonate. The proteins were further digested with 1.0 ⁇ g trypsin (Promega) for 16 h at 37 °C. The digestion was stopped with 0.5 % trifluoracetic acid (TFA) and the peptides desalted on a reversed-phase SepPak CI 8 cartridge (Waters); peptides were eluted using 80% acetonitrile (ACN). The eluate was dried in a SpeedVac and the peptides acidified with 2% ACN-0.1% TFA.
  • TFA trifluoracetic acid
  • Unassigned precursor ion charge states as well as singly charged species were excluded from fragmentation.
  • the isolation window was set to 3 Da and fragmented with a normalized collision energy of 27.
  • the maximum ion injection times for the survey scan and the MS/MS scans were 20 ms and 60 ms, respectively, and the ion target values were set to le6 and le5, respectively.
  • Selected sequenced ions were dynamically excluded for 30 seconds. Data were acquired using Xcalibur software (Thermo).
  • Mass spectra were analyzed using MaxQuant software version 1.5.2.8 using the Andromeda search engine. The initial maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. Enzyme specificity was set to trypsin, defined as C-terminal to arginine and lysine excluding proline, and a maximum of two missed cleavages were allowed. Carbamidomethylcysteine was set as a fixed modification, and N-terminal acetylation and methionine oxidation as variable modifications.
  • the spectra were searched with the Andromeda search engine against the Human and RSV SWISSPROT sequence database (containing 20, 193 human protein entries and 11 RSV protein entries) combined with 248 common contaminants, and concatenated with the reversed versions of all sequences.
  • Protein identification required at least one unique or razor peptide per protein group. Quantification in MaxQuant was performed using the built in XlC-based label-free quantification (LFQ) algorithm (29). The required false discovery rate (FDR) for identification was set to 1% at the peptide and 1% at the protein level, and the minimum required peptide length to 6 amino acids. Contaminants, reverse identification and proteins only identified by site were excluded from further data analysis.
  • the raw data, and database search results are deposited in ProteomeXchange under Project Accession Number PXD005814.
  • the LFQ values were log2-transfomed. After filtering (at least 2 valid LFQ values in at least one group), remaining missing LFQ values were imputed from a normal distribution (width: 0.3; down shift: 1.8). Significance analysis of microarrays (SAM) was used to assess the statistical significance of protein abundances using 1% FDR adjustment and a two-fold cutoff.
  • SAM Significance analysis of microarrays
  • the normalized spectral abundance factor (NSAF) value for each protein was calculated as where the total MS intensity (I) of the matching peptides from protein k was divided by the protein length (L) and then by the sum of I/L for all uniquely identified proteins in the dataset.
  • Stable isotope dilution (SID)-selected reaction monitoring (SRM)-MS Stable isotope dilution (SID)-selected reaction monitoring (SRM)-MS.
  • SID-SRM- MS assays were developed.
  • the peptides were chemically synthesized incorporating isotopically labeled [ 13 C6 15 N 4 ] arginine or [ 13 C 6 15 N 2 ] lysine to a 99% isotopic enrichment (Thermo Scientific).
  • the secretome and cellular proteome were digested as described above.
  • the tryptic digests were reconstituted in 30 ⁇ of 5% formic acid/0.01% TFA.
  • An aliquot of 10 ⁇ of diluted stable isotope-labeled standard (SIS) peptides was added to each tryptic digest.
  • SIS stable isotope-labeled standard
  • each tryptic digest was injected onto a C18 reversed-phase nano-HPLC column (PicoFritTM, 75 ⁇ x 10 cm; tip ID 15 ⁇ ) at a flow rate of 500 nL/min with a 20-min 98% A, followed by a 15-min linear gradient from 2-30% mobile phase B (0.1 %> formic acid/90%) acetonitrile) in mobile phase A (0.1 %> formic acid).
  • the TSQ Vantage was operated in high-resolution SRM mode with Ql and Q3 set to 0.2 and 0.7-Da Full Width Half Maximum (FWHM).
  • All acquisition methods used the following parameters: 1800 V ion spray voltage, a 275°C ion transferring tube temperature, a collision- activated dissociation pressure at 1.5 mTorr, and the S-lens voltage used the values in the S- lens table generated during MS calibration.
  • Quantitative Real Time PCR For gene expression analyses, 1 ⁇ g of RNA was reverse-transcribed using Super Script III in a 20 ⁇ _, reaction mixture (34). One ⁇ _, of cDNA product was diluted 1 :2, and 2 ⁇ _, of diluted product was amplified in a 20 ⁇ _, reaction mixture containing 10 ⁇ _, of SYBR Green Supermix (Bio-Rad) and 0.4 ⁇ each of forward and reverse gene-specific primers. The reaction mixtures were aliquoted into a Bio- Rad 96-well clear PCR plate and the plate sealed with Bio-Rad Microseal B film before insertion into the PCR machine.
  • Q-RT-PCR Quantitative Real Time PCR
  • the plates were denatured for 90 s at 95°C and then subjected to 40 cycles of 15 s at 94°C, 60 s at 60°C, and 1 min at 72°C in an iCycler (Bio- Rad). PCR products were subjected to melting curve analysis to assure that a single amplification product was produced. Quantification of relative changes in gene expression was calculated using the AACt method. Data were expressed as fold change mRNA normalized to cyclophilin or PolB mRNA abundance as indicated as an internal control.
  • BALB/c mice (Harlan) were inoculated intranasally (in) with 50 ⁇ _, of pRSV (final inoculum, 10 7 pfu) diluted in PBS under light anesthesia. Twenty-four hours later, the animals were euthanized and their lungs fixed in paraformaldehyde for immunohistochemical analysis. Sections were prepared and stained with H&E or Periodic Acid Schiff (Abeam) stains using standard techniques.
  • Fig. 17 shows the differential expression of secreted proteins (top) vs cell lysates (bottom) for hBECS, while Fig. 17 (right panel) shows the differential expression of secreted proteins in secreted proteins vs lysates for hSAECs.
  • apoptosis and necrosis rates of control and RSV-infected tert-hBECs and tert-hSAECs were measured by flow cytometry.
  • Cellular necrosis was minimal in both cell types in the absence or presence of RSV infection.
  • tert-hBECs had a higher basal apoptotic rate than did tert-hSAECs (18.8% vs 10.8%).
  • RSV infection reduced the apoptosis rate.
  • tert-hBECs fell from 18.8 to 12.5%
  • tert-hSAECs fell from 10.8% to 7.9%. The data confirms that under these conditions, the cells are viable, and actively replicating and secreting RSV.
  • Secretome proteins are a distinct population of proteins from that produced by cellular lysis
  • control tert-hBEC WCL formed a distinct cluster from that of the tert-hSAEC WCLs, separated by the second principal component dimension.
  • the RSV-infected tert-hBEC WCLs moved in the second dimension to form a cluster that overlapped with both control and RSV-infected tert-hSAEC WCLs.
  • Both the control secretomes of tert-hBECs and tert-hSAECs clustered together, widely separated in the first dimension from the WCL clusters.
  • both the tert-hBEC and tert- hSAEC secretomes migrated up in the second dimension and down in the first dimension.
  • 460 proteins were present only in the secretome, 885 proteins were unique to the cellular proteome, and 1,044 proteins were present in both datasets.
  • GOCC genome ontology cellular component
  • NSAF normalized spectral abundance factor
  • proteins enriched in the CM included macrophage inhibitory factor (MIF-1), macrophage migration CXCL-10, interferon- stimulated gene- 15, high mobility group box (HMGB) 1/2, interferon-induced protein with tetratricopeptide repeats (IFIT)-3, IFN lambda 2, and others.
  • MIF-1 macrophage inhibitory factor
  • HMGB high mobility group box
  • IFIT interferon-induced protein with tetratricopeptide repeats
  • RSV induces exosome production in a cell type -dependent pattern
  • cytosolic proteins may be secreted via unconventional protein secretory pathways, perhaps mediated by Golgi or endosomal export mechanisms.
  • cytosolic proteins may be secreted via unconventional protein secretory pathways, perhaps mediated by Golgi or endosomal export mechanisms.
  • 65 of these common proteins including heat shock cognate 71 kDa protein (HSPA8), glyceraldehyde-3-phosphate GAPDH), and annexin A2 (ANXA2) are prominent exosomal proteins.
  • HSPA8 heat shock cognate 71 kDa protein
  • GAPDH glyceraldehyde-3-phosphate GAPDH
  • ANXA2 annexin A2
  • Ultracentrifuge-purified exosomes manifested 1 12.8 ⁇ 2.0 nm in size by dynamic light scattering, and exhibited a characteristic membrane composition in TEM (Fig. 18). From this fraction, 937 exosomal proteins were identified; of these, 564 proteins were identified in the secretome (373 proteins unique to the exosomal fraction were below the limit of detection and not observed in the secretome analysis). The 937 proteins were subjected to GOCC. Significantly enriched cellular components included lysosomal, vacuolar, and endoplasmic reticular. Out of 937 identified exosome proteins, 853 were quantified across the experimental groups.
  • RSV infection caused up-regulation of 220 tert-hSAEC and 241 tert-hBEC exosomal proteins; and downregulation of 146 tert-hSAEC and 223 tert-hBEC exosomal proteins (FDR ⁇ 0.05).
  • the protein contents in the exosome also display cell type differences. In the basal state, 31 proteins were more abundant in the tert-hSAEC exosome fraction, while 60 proteins were more abundant in the tert-hBEC exosome fraction (FDR ⁇ 0.05).
  • SAM Statistical analysis of microarray was applied to identify differentially expressed proteins using 1% false discovery rate (FDR). SAM identified 71 proteins distinct in the control secretomes from tert-hBECs vs tert-hSAECs. Of these, 61 were upregulated in the tert-hSAEC secretome. Similarly, 131 proteins showed differential expression in the RSV- induced secretomes from tert-hBECs and tert-hSAECS. Of these, 65 were upregulated in hSAECs.
  • the differentially expressed proteins were subjected to 2-dimensional hierarchical clustering.
  • Hierarchical clustering groups proteins whose expression patterns are most similar across cell type and treatment condition. The samples are also clustered by the patterns of proteins. In this analysis, the treatment groups co-clustered in the vertical dimension, consistent with the findings in PC A analysis that the replicates from each cell type are highly similar.
  • PC A analysis that the replicates from each cell type are highly similar.
  • 5 distinct patterns of protein expression emerged.
  • One group represented proteins abundant in control tert-hBECs whose expression is inhibited in response to RSV (not expressed by tert-hSAECs).
  • One group represented proteins expressed by tert-hSAECs whose abundance is decreased in response to RSV.
  • One group represented proteins only expressed by RSV-infected tert-hSAECs.
  • One group represented proteins induced by RSV that are common to both cell types.
  • One group represented proteins induced by tert-hBECs but not tert-hSAECs.
  • One of these groups contained a number of immunologically significant proteins, including CXCL1, IL-6 and CCL20. These data suggest that RSV induces cell-type differences in the secretion of immunologically significant cytokines.
  • our analysis pipeline developed using tert-immortalized human epithelial cells enables the reliable analysis of epithelial secretomes to understand differences by cell type and RSV-induced expression patterns.
  • Secretome fractions were prepared from control and RSV-infected primary cells. We identified 2,376 proteins with FDR 1%. SAM analysis identified 577 proteins in the secretome from control (uninfected) cells whose expression varied by cell type, and 966 proteins in the secretome from RSV-infected cells whose expression varied by cell type (FDR of 1%). To focus on the most robust differentially expressed proteins, we filtered proteins that showed a 5-fold expression change or greater; this resulted in 492 of the most highly significant and induced proteins. This filtered dataset was subjected to 2-dimensional hierarchical clustering, where each column represents a sample and each row represents an individual protein's abundance.
  • cluster 2 represents 1 1 proteins upregulated by RSV infection in phBECs
  • cluster 3 represents 1 16 proteins upregulated by RSV infection in phSAECs
  • cluster 4 contains 203 proteins induced by both cell types (Table II).
  • GSEA Gene Set Enrichment Analysis
  • GSEA identified enrichment of canonical pathways for glycoxylate, carbonyl, porphyrin and amino acid metabolism, and oxidative reduction. This analysis suggests that phSAECs secrete proteins controlling nucleotide-sugar bioenergetic processes in response to RSV.
  • the 203 proteins secreted by both epithelial cell types were analyzed in the same manner. This analysis identified the most GO functions numerically, many of which could be collapsed into mRNA processing (splicing, catabolism, ribonuclease complex assembly), DNA cell cycle regulation and others. GSEA indicated enrichment in mRNA destabilization, Wnt signaling, HIV factor interactions and mRNA interaction/metabolism.
  • Upstream regulator analysis compares the known effect (transcriptional activation or repression) of a transcriptional regulator on its target genes to the observed changes in protein abundance.
  • EHF epithelium-specific ets homologous factor
  • KLK-6/7 kallikrein-related peptidase
  • phSAECs the NFkB transcription factor was predicted to be activated to a greater degree in response to RSV infection than in hBECs.
  • the NFkB network is responsible for regulating TSLP, CCL20, BMP2, MMP3 and SOD2.
  • TSLP is produced by RSV-infected airway epithelium and promotes Th2 differentiation by inducing the maturation of antigen-presenting dendritic cells.
  • CCL20 is a potent inducer of epithelial mucin production, Thl7 lymphocyte and dendritic cell chemotaxis that promotes formation of mucosal lymphoid tissue formation and plays an important role in the pathology of RSV-induced lung inflammation.
  • Chemokine (C-C motif) ligand 3-like 1 (CCL3-L1) is chemotactic for monocytes and lymphocytes, and interacts with CCR5, a receptor linked to RSV LRTI.
  • phSAEC-secreted CCL20 is a biologically active mucin inducer
  • mucins constitute an important arm of the innate immune response via their ability to trap microorganisms
  • mucins also play an important role in the pathogenesis of airway obstruction in RSV LRTI.
  • mucous plugging of the small airways is an important mechanism for atelectasis in bronchiolitis, producing ventilation-perfusion mismatching and hypoxia.
  • phBECs express the CCR6 receptor CCR6 mRNA expression by Q-RT-PCR. Both cell types express CCR6 in uninfected and infected conditions.
  • CCL20 is the major mucin-promoting cytokine in hSAECs secretome, and validate its preferential expression in lower airways in a BALB/c mouse model of RSV infection.
  • Exosomes are small 90-100 nm extracellular vesicles derived from endosomal multivesicular bodies (exosomes) that function in extracellular signal transduction. Enclosed within protective phospholipid bilayers, exosomes proteins,vasoactive leukotrienes, and small RNAs (sncRNA and miRNAs), whose dynamic composition is determined by the microenvironment of the secreting cell. Currently we understand that exosomes participate in signal transduction in distant target cells, affect cellular behavior.
  • exosomal content is affected by changes in the microenvironment of the cellular origin
  • profiling and quantification of exosome content may be used as "liquid biopsies" to detect occult cellular stress, inflammation, tissue injury, wound healing, tissue remodeling and cancer.
  • the Z-average DLS % intensity size distribution metric was compared for the different storage conditions.
  • Freshly prepared BALF exosomes produced an asymmetric size distribution of vesicles from 50-170 nm, with an average size of 94.5 ⁇ 1.7 nm.
  • exosomes stored at 4 °C underwent a size shift of its average size to 104 ⁇ 1.15 nm in three independent isolations (p ⁇ 0.05, t -test).
  • a much more dramatic change was observed for BALF exosomes stored at -80 °C, where the average increased to 125 ⁇ 1.15 nm (p ⁇ 0.001, t-test).
  • Exocarta 80 of the top 100 exosomal proteins in Exocarta were identified, establishing that this preparation was enriched in exosomes. Functional analysis showed that these proteins were significantly enriched in 39 biological pathways, including tRNA aminoacylation, glycolysis, translation, macrophage activation, proteolysis and others (Table IV).
  • BALF exosomes undergo dynamic changes in protein (cytokines), arachidonic acid (leukotrienes) and miRNA content that could potentially regulate innate immunity, hyper- responsiveness and cellular inflammation in the airway.
  • cytokines protein
  • arachidonic acid leukotrienes
  • miRNA content could potentially regulate innate immunity, hyper- responsiveness and cellular inflammation in the airway.
  • our study was not designed to examine the effect of poly(LC) inflammation on exosomal content, our data significantly extends the number of proteins contained within BALF exosomes.
  • 848 high confidence proteins that are enriched in 80 of the top 100 proteins in the ExoCarta database that have previously been identified as exosomal markers.
  • Pathway analysis indicates that the poly(LC) induced exosomes are enriched in a number of biological processes. These enriched biological processes include tRNA aminoacylation and protein translation (Table IV), perhaps participating in the processing or activity of the miRNA content.
  • Table V GO analysis of peri-exosomal proteins. 699 high confidence peri-exosomal proteins were analyzed for biological processes by GO-Slim (Panther database). Shown is fold enrichment of the pathway and p value (bonferroni correction).
  • RNA metabolic process (GO: 0016070) 0.53 1.81E-02 developmental process (GO:0032502) 0.5 7.71E-03 cell surface receptor signaling pathway (GO: 0007166) 0.42 4.02E-03 neurological system process (GO:0050877) 0.34 4.45E-05 transcription from RNA polymerase II promoter (GO: 0006366) 0.27 2.92E-04 regulation of transcription from RNA polymerase II promoter
  • G-protein coupled receptor signaling pathway (GO:0007186) 0.25 1.22E-02

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Abstract

L'invention concerne des procédés et des dispositifs pour la détection microfluidique d'un marqueur biologique dans un échantillon biologique collecté d'un sujet. Les dispositifs microfluidiques comprennent des nanocapteurs à base de nanoparticules comprenant des séquences supramoléculaires de reconnaissance, des séquences consensus de protéases, des séquences modifiables post-traduction, ou des liaisons benzyléther encombrées stériquement pour une interaction spécifique avec un marqueur biologique. L'invention concerne également des nanocapteurs particuliers permettant de détecter des cytokines, et d'autres protéines sur la base d'une reconnaissance supramoléculaire sans modification chimique ni clivage enzymatique.
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